Prevention of leaching of ligands from affinity-based purification systems

ABSTRACT

In an affinity-type purification, ligands dissociated from the stationary phase that would otherwise leach into the species being purified are captured by a second ligand that is also incorporated into the stationary phase, the second ligand exhibiting an affinity-type interaction with the dissociated first ligand with sufficient specificity to avoid the undesired retention by the second ligand of species from the liquid sample or source liquid other than the species sought to be purified in the affinity column.

CROSS REFERENCE TO RELATED APPLICATION

This application is as division of co-pending application Ser. No.11/563,961, filed Nov. 28, 2006, the contents of which are incorporatedherein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention resides in the field of affinity chromatography, andaddresses concerns arising from the labile character of ligands coupledto a support as the stationary phase.

2. Description of the Prior Art

Affinity chromatography is widely used for separating and detectingcomponents in biological samples and for the isolation or purificationof biological species or recombinant species from clinical samples, fromcell growth cultures, or from any medium in which the species areproduced or can be extracted. Affinity chromatography is commonlyperformed by passing a liquid medium containing the species of interestthrough a column or membrane to which a ligand is bound as a stationaryphase, the ligand being one to which the species of interest binds by anaffinity-type interaction. Affinity chromatography that is used toisolate and purify species is also termed “affinity extraction,” and thespecies-ligand interaction in this type of extraction is one that occurswith sufficient specificity to differentiate between the species ofinterest and other species in the source liquid. Affinity extractiontechniques include immunoextraction in which the ligands are antibodies;protein-protein extractions using such ligands as wheat germ agglutinin,concanavalin A, protein A, and protein G; and interactions involvingnon-protein species such as heparin or nucleic acids. Once immobilizedon the solid phase by the affinity interaction, the species of interestis removed from the solid phase by an appropriate change in conditionssuch as a change in pH or the introduction of a detergent, chaotrope,salt, competitive binding species, or any agent that will overcome orlessen the binding affinity of the species to the solid phase. The typesof changes that will be effective in releasing the bound species inparticular systems are well known in the art of affinity chromatography.

The ligand is typically a protein or other affinity-binding species thatis coupled to a solid support by covalent bonds, the support oftenhaving been activated to facilitate the formation of such bonds.Activation commonly involves the placement of a reactive group, such asfor example an epoxide group, on the support surface. The linkagebetween the ligand and the support is labile, however, often resultingin dissociation of the ligand from the support during the passage ofliquids through the medium. In addition, leaching can occur by enzymaticor chemical degradation of the ligand itself, such as proteolysis ofprotein affinity ligands by proteases in the process stream, cleavage ofnucleic acids by endo- and exo-nucleases in the process stream, etc. Theaffinity ligand that is leached from the affinity medium as a result ofthis dissociation may be small compared to the amount of ligandremaining on the support, but even a small amount of leached ligand canseriously contaminate the otherwise purified species eluted from themedium. When a therapeutic agent that is either biologically derived orproduced by recombinant chemistry is contaminated with a leachedaffinity ligand, the leached ligand can recombine with the agent andthereby impede the effectiveness of the agent, or combine with, orimpede the functions of, other species such as membranes, cell walls, orenzymes in the patient's body to cause harm. Concavalin A, for example,is an affinity ligand that is used for purifying lysosomal enzymepreparations, but is known to leach from affinity columns andcontaminate the enzyme preparations, particularly by activating T cellsin the patient to whom the enzyme preparation is administered. Toeliminate these types of contamination, the leached ligands must beremoved, and this is typically performed by separations downstream ofthe affinity column or membrane. This adds cost and time to thepreparation.

SUMMARY OF THE INVENTION

The present invention resides in an affinity medium, method, and systemthat prevents species purified in the medium from being contaminatedwith leached proteins from the medium or leached segments of theproteins that have become dissociated during the passage of liquidsthrough the medium. This is achieved by a stationary phase that containsat least two ligands immobilized on a support. The first ligand is theligand that binds to the solute, i.e., the species to be purified, andthe second ligand is one that binds selectively to molecules of thefirst ligand or dissociated segments thereof that are not, or are nolonger, immobilized on the support. Both ligands bind to theirrespective binding partners by an affinity-type interaction. Thus, whilethe affinity-binding proteins are initially immobilized on thestationary phase by covalent binding, leached molecules of theseproteins are captured by affinity binding in the same separation medium,whether the medium be a column, a membrane, or any other form ofstationary phase. A single affinity medium therefore serves both toisolate the species of interest and to remove contaminants that wouldotherwise arise within the affinity medium itself.

These and other features, advantages, and objects of the invention willbe apparent from the description that follows.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

Ligands used as the stationary phase in the practice of this inventionare referred to herein as the “first ligand” to differentiate theseligands from the “second ligand” which represents ligands used tocapture leached molecules of the first ligand. The first ligand includesany of the wide variety of ligands that are used in affinitychromatography, and preferably those that are disclosed in theliterature or used in clinical laboratories, research laboratories, orproduction facilities, as stationary phases for affinity extraction.First ligands can be protein ligands, polysaccharides, or other largemolecules that engage in affinity binding. Lectins are examples ofligands useful as the first ligand, effective for extracting certaintypes of carbohydrates, such as polysaccharides, glycoproteins, andglycolipids. Specific lectins include concavalin A, wheat germagglutinin, jacalin, and lectins found in peas, peanuts, and soybeans.Protein A and protein G, useful in binding to the constant regions ofmany types of immunoglobulins, are further examples of ligands useful asthe first ligand. A ligand demonstrating the binding behavior of bothprotein A and G is the recombinant protein known as protein A/G, whichis also useful as the first ligand. In immunoextraction, as noted above,the first ligand is an antibody (including monoclonal antibodies) or anantibody fragment. Examples of species purified by immunoextractionusing these ligands are anti-idiotypic antibodies, glucosaccharides,granulocyte colony-stimulating factor, human serum albumin, IgG, IgE,interferon, tumor necrosis factor, interleukins, recombinant FactorVIII, and transferrin. Still further examples of the first ligand arenon-protein ligands. Examples of these are aptamers and heparin.Aptamers exhibit antibody-type interactions and are known foraffinity-type binding to adenosine and for chiral separations, whileheparin is useful for purifying certain lipoproteins.

The support for the stationary phase can be of any material andconfiguration that are known to be functional in affinitychromatography. Examples are particles and beads, which can be packed,fluidized, or immobilized. Other examples are fibers, foams, frits,microporous films, membranes and substrates with microreplicatedsurfaces. When a microporous film is used, it can be attached to theinner surface of a capillary, a well in a multi-well plate, or areaction vessel, as a lining on the surface. When particles or beads areused, they can be rigid solids or semi-solids such as gels.

The second ligand, which captures dissociated molecules of the firstligand to prevent these molecules from leaching into the product, ischosen for its affinity binding specificity toward the first ligand, andthe choice will therefore be governed or dictated by the first ligand.Examples of species suitable for use as the second ligand thus includemonoclonal antibodies, proteins, small peptides, aptamers, and organicspecies such as triazines and boronates. The second ligand will beselected as one that does not bind other species in the liquid mixturethat contains the species of interest and thereby does not significantlychange the purity of the final product by virtue of retaining extraneousspecies in the affinity medium, other than molecules of the first ligandthat have become dissociated and would otherwise leach out of themedium.

The first and second ligand can be coupled to the support byconventional coupling chemistries, typically using activated orfunctionalized supports, such as for example epoxide-functionalizedsupports. Examples of the types of linkage are ether linkages, thiollinkages, amino linkages, carboxyl linkages, and aldehyde linkages. Whenthe stationary phase is a non-moving phase, such as a packed bed,membrane, or surface, the first and second ligands can be uniformlydistributed throughout the phase, or they can be fully or partiallysegregated, such as for example with the second ligand concentrated atsites downstream from the first ligand in the direction of the flow ofthe liquid through or across the stationary phase. A uniformdistribution of both ligands throughout the stationary phase ispreferred. When the stationary phase is in the form of particles orbeads, the two ligands can be coupled to separate populations ofparticles or beads and the two populations then mixed together, or thetwo ligands can be coupled to the same particles or beads, such thatcertain particles or beads contain both types of ligand.

The relative amounts of first and second ligand can be selected on thebasis of known or suspected dissociation rates of the first ligand, andmay vary with the first ligand and the type of linkage joining the firstligand to the support. In most cases, however, best results will beachieved when the molar percentage of second ligand relative to firstligand is from about 0.01% to about 30%, and preferably from about 0.3%to about 10%. In terms of the weight percent of the ligands relative toa resin that serves as the support, the amount of first ligand willoften be from about 0.3 g to about 30 g of ligand per liter of resin, ormore often from about 1 g to about 10 g per liter of resin, and theamount of second ligand will often be from about 0.001 g to about 10 gper liter of support, or preferably from about 0.003 g to about 3 g perliter of support.

A source liquid containing a solute of interest can be placed in contactwith an affinity medium meeting the above description for purposes ofpurification in the same manner that such liquids are contacted withaffinity media of the prior art. The medium can thus be retained in aflow-through column and a source liquid passed through the column, orthe medium can be suspended in the source liquid, incubated sufficientlyto allow the binding to occur, and the solid and liquid phases thenseparated. In either case, the contact between the source liquid and theaffinity medium is performed under conditions that will allow the soluteto bind to the affinity medium. Such conditions are likewise known inthe art, and involve such parameters as the pH, ionic strength, contacttime, and the presence or absence of other components in the liquidphase. Once binding has occurred, the unbound components are washed fromthe medium, using conventional washing media that will not causedissociation of the solute of interest. Once the washing is complete,the solute is dissociated from the medium and collected by exposing themedium to the dissociation conditions most appropriate to the speciesinvolved. As noted above, the dissociation conditions may be a change inpH or the introduction of a detergent, a chaotrope, a salt, orcompetitive binding species. The result will be a solution of the solutethat is purified relative to other solutes in the source liquid. Theexpression “purified relative” is used herein to mean that while theconcentration of the solute of interest as recovered from the affinitymedium may be the same, greater than, or less than its concentration inthe source solution, other solutes originally present in the sourcesolution will be either significantly reduced in concentration, reducedto concentrations below the level of detection, or eliminated entirely.

The terms “a” or “an” as used in the appended claims are intended tomean “one or more.” The term “comprise,” and variations thereof such as“comprises” and “comprising,” when preceding the recitation of a step oran element is intended to mean that the addition of further steps orelements is optional and not excluded. All patents, patent applications,and other published reference materials cited in this specification arehereby incorporated herein by reference in their entirety. Anydiscrepancy between any reference material cited herein and an explicitteaching of this specification is intended to be resolved in favor ofthe teaching in this specification. This includes any discrepancybetween an art-understood definition of a word or phrase and adefinition explicitly stated in this specification of the same word orphrase.

The foregoing is offered primary for purposes of illustration. Furthervariations and substitutions that likewise utilize the fundamentalfeatures of novelty and utility described herein will be apparent tothose skilled in the art and are likewise encompassed by the scope ofthe claims.

1. An affinity medium for extracting a solute from a liquid sample, saidaffinity medium comprising first and second ligands immobilized on acommon membrane, said first ligand binding selectively to said solute byaffinity-type binding, and said second ligand binding selectively byaffinity-type binding to molecules of said first ligand or to segmentsof said molecules upon release of said molecules or segments from saidmembrane.
 2. The affinity medium of claim 1 wherein said first andsecond ligands are both immobilized on said membrane by covalent bindingto said membrane.
 3. The affinity medium of claim 1 wherein said firstligand is a member selected from the group consisting of a lectin,heparin, protein A, and protein G.
 4. The affinity medium of claim 1wherein said second ligand is a member selected from the groupconsisting of a monoclonal antibody, an aptamer, a triazine, and aboronate.
 5. The affinity medium of claim 1 wherein said second ligandis present on said medium in a quantity equal to 0.01% and about 30% ofsaid first ligand, on a molar basis.
 6. The affinity medium of claim 1wherein said second ligand is present on said medium in a quantity equalto 0.3% and about 10% of said first ligand, on a molar basis.
 7. Aprocess for extracting a solute from a liquid sample, said processcomprising (a) contacting said liquid sample with an affinity medium inaccordance with claim 1, under conditions causing said solute to bind tosaid first ligand, (b) washing said affinity medium of said sample whilesaid solute is so bound, and (c) dissociating said solute from saidaffinity medium in a purified form relative to other solutes in saidsample.
 8. The process of claim 7 wherein said first and second ligandsare both immobilized on said membrane by covalent binding to saidmembrane.
 9. The process of claim 7 said first ligand is a memberselected from the group consisting of a lectin, heparin, protein A, andprotein G.
 10. The process of claim 7 wherein said second ligand is amember selected from the group consisting of a monoclonal antibody, anaptamer, a triazine, and a boronate.